Autologous thymic tissue transplantation

Abstract

To provide autologous thymic tissue transplantation.SOLUTION: The present disclosure provides methods and kits for preserving or restoring thymic functions of a subject in need thereof. The methods and kits disclosed herein include a step of delivering autologous thymic tissue into at least one lymph node of the subject. In one aspect, the present disclosure provides a method for preserving or restoring thymic functions of a subject that has received or is scheduled to receive a thymectomy surgery. The present invention provides, for example, a method for preserving or restoring thymic functions of a subject in need thereof, the method comprising a step of delivering thymic tissue into at least one lymph node of the subject, wherein the thymic tissue is autologous to the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 882,887, filed August 5, 2019, the entire contents of which are incorporated herein by reference.

[0002] 2.Technical Field The present disclosure provides methods of protecting or restoring thymic function in a subject (eg, a human subject) in need thereof, for example, after thymectomy. [Background technology]

[0003] 3.Background The thymus is a lymphoid organ of the immune system that can produce and mature T cells. Thymectomy, a surgical removal of the thymus, is often performed to treat various thymus-related disorders. Thymectomy can be an effective treatment option for patients with myasthenia gravis, thymoma, or thymic tumors.

[0004] The thymus is known to be most active during fetal life and early postnatal life, and its size and activity may begin to decline after birth. Thymectomy may be routinely performed in certain children (e.g., newborns and infants) to repair congenital heart defects. Because the thymus may be located below the sternum, blocking the pathway to the heart, partial or total thymectomy can facilitate access to and visualization of the heart and great vessels. In the United States, approximately 20,000 infants undergo thymectomy each year.

[0005] However, thymectomy is thought to eliminate the primary source of T cell maturation and can lead to severe long-term clinical consequences, such as autoimmune disease, cancer, infection, atopic disease, and premature immunological aging. Despite its frequent use, no fully effective treatments are available for thymectomized children.

[0006] Thus, there remains a need for effective treatments to protect or restore thymic function in thymectomized subjects. The inventive subject matter disclosed herein provides such treatments. Summary of the Invention [Means for solving the problem]

[0007] 4. Summary of the Invention The present disclosure provides techniques and kits for protecting or restoring thymic function in a subject (e.g., a human subject) in need thereof. The subject matter disclosed herein is based, at least in part, on the discovery that children, particularly newborns and infants, who undergo thymectomy can develop serious long-term clinical disorders and diseases, including autoimmune diseases, cancer, infectious diseases, atopic diseases, and immunological premature aging.

[0008] In one aspect, the present disclosure provides a method of protecting or restoring thymic function in a subject who has undergone or will undergo thymectomy, the method comprising delivering thymic tissue into at least one lymph node of the subject, wherein said thymic tissue is autologous to said subject.

[0009] In certain embodiments, the subject has a congenital heart defect, hi certain embodiments, the subject has undergone or is scheduled to undergo open heart surgery.

[0010] In certain embodiments, the subject has undergone or will undergo thymectomy. In non-limiting embodiments, the thymectomy was performed when the subject was a newborn or infant.

[0011] In certain embodiments, the subject is a human subject. In certain embodiments, the subject is a newborn or infant. In certain embodiments, the subject is a child, adult, or adolescent.

[0012] In certain embodiments, the thymus tissue is obtained from a subject undergoing a thymectomy. In certain embodiments, the thymus tissue is obtained from a subject prior to a thymectomy.

[0013] In certain embodiments, the thymus tissue is a minced thymus slice. In certain embodiments, the thymus tissue is cultured ex vivo before delivery. In certain embodiments, the thymus tissue is cultured ex vivo for at least 24 hours before delivery. In certain embodiments, the thymus tissue is delivered into the lymph nodes of the subject by needle.

[0014] In certain embodiments, the thymus tissue is delivered into a lymph node of the subject by needle.

[0015] In certain embodiments, thymus tissue is delivered into the lymph nodes during a thymectomy, hi certain embodiments, thymus tissue is delivered into the lymph nodes after a thymectomy.

[0016] In certain embodiments, the thymus tissue is cryopreserved before being delivered into the lymph node.In certain embodiments, the cryopreserved thymus tissue is delivered into the lymph node after thymectomy.In certain embodiments, the cryopreserved thymus tissue is delivered into the lymph node about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 4 months, about 6 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, about 15 years, about 20 years or more after thymectomy.

[0017] In certain embodiments, the amount of thymic tissue is effective to restore thymic function in a subject, hi certain embodiments, the amount of thymic tissue is effective to expand in a lymph node, wherein the expanded thymic tissue restores thymic function in a subject.

[0018] In certain embodiments, the amount of thymus tissue is at least about 0.1 grams. In certain embodiments, the amount of thymus tissue is up to about 20 grams. In certain embodiments, the size of the thymus tissue is at least about 0.1 cm3 In certain embodiments, the size of the thymus tissue is about 20 cm 3 That's it.

[0019] In certain embodiments, the thymus tissue is delivered into at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten lymph nodes of a subject. In certain embodiments, the thymus tissue is cryopreserved thymus tissue. In non-limiting embodiments, at least about one-third of the subject's total thymus weight is delivered to at least one lymph node of the subject.

[0020] In another aspect, the present disclosure provides a kit for protecting or restoring thymic function in a subject, the kit comprising thymic tissue and tools for delivery of said thymic tissue to at least one lymph node of the subject, wherein said thymic tissue is autologous to said subject.

[0021] In certain embodiments, the kit further comprises a solution, wherein the thymus tissue is provided in the solution, hi certain embodiments, the solution comprises a pharmaceutically acceptable excipient, a pharmaceutically acceptable diluent, or a pharmaceutically acceptable carrier.

[0022] In certain embodiments, the amount of thymic tissue is effective to restore thymic function in a subject, hi certain embodiments, the amount of thymic tissue is effective to expand in a lymph node, wherein the expanded thymic tissue restores thymic function in a subject.

[0023] In certain embodiments, the kit further comprises instructions for delivering thymus tissue to at least one lymph node of the subject. In non-limiting embodiments, the instructions comprise delivering at least about one-third of the subject's total thymus weight into at least one lymph node of the subject.

[0024] In certain embodiments, tools for delivery of thymus tissue include needles and tools necessary for minimally invasive procedures.

[0025] In certain embodiments, the subject has undergone or will undergo a thymectomy. 5. Brief description of the drawings [Brief explanation of the drawings]

[0026] [Figure 1A] 1A-1C illustrate harvested and transplanted wild-type newborn thymuses. FIG. 1A shows a bar graph of the weight of BALB / c wild-type newborn thymuses used for transplantation of the present disclosure. FIG. 1B shows a bar graph of the number of BALB / c nude mice transplanted with a quarter lobe, a half lobe, a full lobe, or a whole thymus of the present disclosure. FIG. 1C shows a bar graph of the number of BALB / c nude mice transplanted with each dose of thymus, expressed in mg, of the present disclosure. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 2A] Figures 2A-2E show flow cytometry analysis of peripheral blood T cells 4 months after transplantation. Figure 2A shows flow cytometry dot plots of the percentage of CD3+ T cells in untransplanted (UnTx) BALB / c nude mice, thymus-transplanted (Tx) BALB / c nude mice, and wild-type BALB / c mice of the present disclosure. Cells were gated first for singlets, then live cells, CD45+ cells, and finally for CD3 expression. Figure 2B shows a scatter plot of the mean percentage of CD45+ cells that are CD3+ according to the present disclosure. Figure 2C shows a scatter plot of the mean percentage of CD3+ cells that are CD4+ according to the present disclosure. Figure 2D shows a scatter plot of the mean percentage of CD3+ cells that are CD8+ according to the present disclosure. Figure 2E shows a scatter plot of the mean and SEM of the percentage of CD45+ cells that are CD3+ T cells in BALB / c nude mice transplanted with a thymus according to the present disclosure. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 3A] Figures 3A-3B show flow cytometry analysis of peripheral blood T cells 5 months after transplantation. Figure 3A shows a bar graph with the mean and standard deviation of the percentage of CD45+ cells that are CD3+ T cells in BALB / c nude mice transplanted with a quarter lobe, half lobe, one lobe, or two lobes of the thymus of the present disclosure. Figure 3B shows a bar graph of the percentage of T cells expressing TCR-Vβ variants in mice as in Figure 3A. BALB / c wild-type mice serve as a positive control (CNTRL). [Figure 3B] Same as above. [Figure 4A] Figures 4A-4B show lymph node weights 6 months after transplantation of thymic tissue fragments. Figure 4A shows a scatter plot of the average lymph node (LN) weights in BALB / c nude mice transplanted with the thymic lobes of the present disclosure. Figure 4B shows a scatter plot of the average lymph node (LN) weights and SEM in BALB / c nude mice transplanted with the thymus of the present disclosure. [Figure 4B] Same as above. [Figure 5A]Figures 5A-5H show flow cytometry dot plots illustrating gating approaches using flow cytometry for effector / effector memory T cells (CD4+CD3+CD44+CD62L-) and (CD8+CD3+CD44+CD62L-); central memory T cells (CD4+CD3+CD44+CD62L+) and (CD8+CD3+CD44+CD62L+); activated effector T cells (CD4+CD3+CD44-CD62L-) and (CD8+CD3+CD44-CD62L-); and naive T cells (CD4+CD3+CD44-CD62L+) and (CD8+CD3+CD44-CD62L+). Figure 5A shows a forward scatter gate versus a side scatter gate. Figure 5B shows a gate for single cells. Figure 5C shows a gate for live cells. Figure 5D shows gating for CD45+ cells. Figure 5E shows gating for CD3+ cells. Figure 5F shows gating for CD4+ cells and CD8+ cells. Figure 5G shows gating for CD4+ cells for naive cells, activated effector cells, effector memory cells, and central memory cells. Figure 5H shows gating for CD8+ cells for naive cells, activated effector cells, effector memory cells, and central memory cells. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 5H] Same as above. [Figure 6A]Figures 6A-6E show flow cytometry analysis of peripheral blood T cell subsets 7 weeks after transplantation of thymic tissue fragments into lymph nodes of adult and aged mice. Figure 6A shows flow cytometry dot plots of CD4+ naive, effector, and effector memory T cell subpopulations, and central memory T cell subpopulations in adult or aged C57BL / 6J female mice with or without transplantation (Tx) of a quarter thymic lobe of the present disclosure. Figures 6B-6E show scatter plots with mean and SEM of the percentage of CD4+ T cells that are activated CD4+ T cell effectors (Figure 6B), CD4+ effector memory T cells (Figure 6C), CD4+ central memory T cells (Figure 6D), and CD4+ naive T cells (Figure 6E) in adult or aged C57BL / 6J female and male mice with or without transplantation of a quarter thymic lobe of the present disclosure. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0027] 6. Detailed Description Non-limiting embodiments of the present disclosure are illustrated herein and by way of example. For clarity, and not by way of limitation, the detailed description is divided into the following subsections: 6.1 Definitions; 6.2 Treatment methods; and 6.3 Kits.

[0028] 6.1 Definition The terms used herein generally have their ordinary meanings within the art, within the context of the disclosed inventive subject matter, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere herein to provide further guidance to the practitioner in describing the compositions and methods of the disclosed inventive subject matter and how to make and use them.

[0029] As used herein, the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or herein, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." Furthermore, the terms "having," "including," "containing," and "comprising" are interchangeable, and those skilled in the art will recognize that these terms are open-ended terms.

[0030] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within 3 or more standard deviations, according to practice in the art. Alternatively, "about" can mean within 20%, preferably 10%, more preferably 5%, and even more preferably 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0031] As used herein, an "individual" or "subject" refers to a vertebrate, such as a human or non-human animal (e.g., a mammal). Mammals include, but are not limited to, humans, non-human primates, livestock, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents (such as mice, rats, hamsters, and guinea pigs); rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates (such as apes and monkeys).

[0032] An adult human subject is a subject who has reached at least about 18 years of age. An adult non-human subject is a subject who has reached sexual maturity. In certain embodiments, a newborn is a human subject who has reached at most about 1 month of age. In certain embodiments, an infant is a human subject who has reached an age between about 1 month and about 2 years of age. In certain embodiments, a child is a human subject who has reached an age between about 2 years and about 12 years of age. In certain embodiments, an adolescent is a human subject who has reached an age between about 12 years and about 18 years of age.

[0033] As used herein, the term "disease" refers to any condition or disorder in which the normal function of a cell, tissue, or organ is damaged or disrupted.

[0034] An "effective amount" of a substance, as that term is used herein, is an amount sufficient to obtain beneficial or desired results, including clinical results, and as such, "effective amount" will depend on the context in which the term is applied. An effective amount can be administered in one or more doses.

[0035] As used herein, and as is well understood in the art, "treatment" refers to an approach for obtaining beneficial or desired results, including clinical results. For purposes of this subject matter, beneficial or desired clinical results include, but are not limited to, reduction or amelioration of one or more signs or symptoms, reduction in the extent of disease, stabilized (i.e., not worsening) disease state, prevention of disease, delay or slowing of disease progression, and / or amelioration or palliation of disease state. A reduction can be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% reduction in the severity of a complication or symptom. "Treatment" can also mean a prolongation of survival compared to the expected survival if not receiving treatment. In certain embodiments, the subject matter disclosed herein is used to restore thymic function in a subject (e.g., a human subject, neonate, infant, child, adolescent, or adult). In certain embodiments, the subject matter disclosed herein is used to increase the level of circulating T cells (eg, naive T cells in peripheral blood) in a subject.

[0036] As used herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, shall be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer).

[0037] As used herein, the term "pharmaceutically acceptable" can refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of a subject (e.g., a human subject or non-human animal) without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0038] As used herein, the term "pharmaceutically acceptable excipient" can refer to a pharmaceutically acceptable material, composition, or vehicle involved in the delivery or transport of a subject compound, material, or cell to an organ or part of the body, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or material encapsulating a solvent, etc. Each excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation (e.g., cells to be transplanted) and not harmful to the subject.

[0039] 6.2 Treatment method The present disclosure provides a method for protecting or restoring thymic function in a subject in need thereof. The method includes delivering thymic tissue (e.g., autologous thymic tissue) into at least one lymph node of the subject to protect, increase, or restore thymic function in the subject. In certain embodiments, the methods disclosed herein can prevent or reduce the short- or long-term clinical effects of thymectomy (such as autoimmune disease, cancer, infectious disease, atopic disease, immunological premature aging, or a combination thereof).

[0040] 6.2.1 Thymus tissue In certain embodiments, the subject has undergone or is scheduled to undergo thymectomy. In certain embodiments, the autologous thymus tissue delivered to the subject is obtained from a subject undergoing thymectomy. In certain embodiments, the autologous thymus tissue delivered to the subject is obtained from a subject prior to thymectomy. In certain embodiments, the thymectomy was performed when the subject was a newborn, infant, or child.

[0041] In some embodiments, the thymus tissue delivered to the subject is not autologous (e.g., allogeneic). In some embodiments, the thymus tissue is derived from a donor who is matched to the subject by one or more human leukocyte antigen (HLA) alleles (e.g., one or both copies of one, two, three, four, five, or six HLA alleles). The thymus tissue can be derived from a donor who is partially HLA-matched, fully HLA-matched, or haploidentical to the subject. To reduce the likelihood of graft rejection, the subject can be placed on an immunosuppressive regimen. Non-limiting examples of drugs that can be administered as part of an immunosuppressive regimen include mTOR inhibitors (e.g., rapamycin), CD28-B7 inhibitors (e.g., CTLA4-Ig), CD40-CD40L inhibitors (e.g., MR1), steroids (such as corticosteroids, dexamethasone, and prednisone), Cox-1 and Cox-2 inhibitors, macrolide antibiotics (such as rapamycin and tacrolimus), cyclosporine, azathioprine, Atgam, thymoglobulin, OKT3, basiliximab, solumedrol, daclizumab, mycophenolate mofetil, prograf, and other substances that limit, reduce, or suppress B cell, T cell, and / or other innate immune activity. An immunosuppressive regimen can include one drug or any combination of suitable drugs administered to achieve the desired effect.

[0042] Thymic tissue for delivery can be prepared using any suitable technique known in the art, including, but not limited to, those described in Market et al., Blood 102, 1121-1130 (2003), the entire contents of which are incorporated herein by reference. In certain embodiments, thymic tissue harvested from a subject is processed (e.g., minced or sliced) into small fragments and resuspended in a liquid to form a solution for delivery into lymph nodes. In some embodiments, grafts containing thymic fragments are superior to single-cell suspension thymic grafts in generating functional ectopic thymuses. For example, grafts containing thymic fragments can increase the amount of thymic tissue engraftment, increase the proportion or number of circulating T cells, increase the proportion or number of circulating naive T cells, or favor other parameters disclosed herein. The solution can preferably be sterile. The solution can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thyromesal, and the like.

[0043] In certain embodiments, the solution contains different cell types that make up thymus tissue. In certain embodiments, the solution further comprises a pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutically acceptable carriers and diluents include saline, buffers, solvents, and / or dispersion media. Non-limiting examples of pharmaceutically acceptable excipients that can be used with the methods of the present disclosure include: sugars (such as lactose, glucose, and sucrose); starches (such as corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate); powdered tragacanth; malt; gelatin; lubricants (such as magnesium stearate, sodium lauryl sulfate, and talc); cocoa butter and suppository wax; oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil). ); glycols (such as propylene glycol); polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG)); esters (such as ethyl oleate and ethyl laurate); agar; buffers (such as magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH-adjusted buffers; polyesters, polycarbonates, and / or polyanhydrides; bulking agents (such as polypeptides and amino acids); serum components (such as serum albumin, HDL, and LDL); C2-C12 alcohols (such as ethanol); and other non-toxic compatible substances used in pharmaceutical formulations.

[0044] In certain embodiments, thymus tissue is cultured ex vivo before being processed and delivered to a subject. Any culture medium known in the art for organ culture can be used with the methods of the present disclosure. In certain embodiments, the medium contains nutrients to maintain tissue viability, at least one antibiotic to prevent microbial infection of the thymus tissue, and a buffer system to maintain the appropriate pH range of the medium. In certain embodiments, the medium contains F12 nutrient mixture, 25 mM HEPES, 2 mM L-glutamine, 10% fetal bovine serum, 100 μg / mL streptomycin sulfate, 1 μg / mL gentamicin, and 100 μg / dL amphotericin B. In certain embodiments, the thymus tissue is cultured in the medium at 37° C. in a 5% CO incubator. In certain embodiments, the thymus tissue is cultured ex vivo for at least about 2 hours, about 4 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 10 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks or more before the thymus tissue is processed or delivered to a subject. In certain embodiments, the thymus tissue is cultured before cryopreservation, after cryopreservation, or a combination thereof. In certain embodiments, the thymus tissue is not cultured ex vivo before processing and / or delivery to a subject.

[0045] In certain embodiments, thymus tissue is cryopreserved before processing or delivery to a subject. Any cryopreservation method for thymus tissue known in the art can be used with the methods disclosed herein (for example, but not limited to, the method disclosed in Jang et al., Integr Med Res. 2017 Mar;6(1):12-18, the entire contents of which are incorporated herein by reference). In certain embodiments, thymus tissue is cryopreserved for at least about 12 hours, about 24 hours, about 2 days, about 3 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, or more before delivery to a lymph node. In certain embodiments, the cryopreserved thymus tissue is delivered into the lymph node long after thymectomy, hi certain embodiments, the thymus tissue is not cryopreserved prior to processing and / or delivery into the subject.

[0046] In certain embodiments, the thymus tissue is delivered into the subject during thymectomy.In certain embodiments, the thymus tissue is delivered into the subject after thymectomy.In certain embodiments, the thymus tissue is delivered into the subject about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years or more after thymectomy. In a non-limiting embodiment, thymus tissue is delivered into a subject during a thymectomy.

[0047] In certain embodiments, the amount of thymic tissue delivered to a subject is an amount effective to restore thymic function in the subject. In certain embodiments, the amount of thymic tissue delivered to a subject is an amount effective to increase thymic function in the subject. In certain embodiments, the amount of thymic tissue delivered to a subject is an amount effective to expand in a lymph node, for example, where the expanded thymic tissue restores thymic function in the subject. In certain embodiments, the amount of thymic tissue delivered to a subject is an amount effective to engraft in a lymph node. In certain embodiments, the amount of thymic tissue delivered to a subject is an amount effective to form ectopic thymic tissue in a lymph node. In certain embodiments, the amount of thymic tissue delivered to a subject is an amount effective to increase the level of circulating T cells (e.g., T cells and / or naive T cells in peripheral blood) in the subject. In certain embodiments, the amount of thymus tissue delivered to a subject is effective to increase the density of circulating T cells in the subject (e.g., increase the TCR repertoire and recognized alloantigens). The effective amount can vary depending on the subject's medical history, age, condition, sex, and the severity and type of the condition in the subject, as well as the administration of other pharmaceutically active agents.

[0048] In certain embodiments, the methods and systems are used to deliver thymocytes or thymus fragments into a subject's lymph nodes, thereby allowing the thymus cells or fragments to engraft in the lymph nodes and produce an ectopic thymus. In certain embodiments, the ectopic thymus restores thymus function in a subject (e.g., complements or enhances one or more functions that a normal, healthy thymus organ can perform). For example, but not by way of limitation, the ectopic thymus may participate in the body's immune regulation by participating in the growth, development, maturation, and selection of T cells. The production of an ectopic thymus of the present disclosure can be used to enhance or regulate immune system function in subjects with impaired immune system function, such as subjects who have undergone thymectomy (e.g., partial or complete removal of the thymus).

[0049] In certain embodiments, the present disclosure relates to the transplantation of thymus tissue from a newborn or infant subject into the subject's own lymph nodes. In some embodiments, the subject receives the transplant at the time the thymus tissue is obtained from the body or shortly thereafter (e.g., within 24 hours, within 1 week, within 1 month, or within 1 year). In some of these embodiments, the subject is a newborn or infant at the time of receiving the transplant into the lymph node. In some embodiments, the subject receives the transplant multiple years after the thymus tissue was removed from the subject. For example, the thymus tissue was removed from the subject when the subject was a newborn or infant, but the subject is an adult, child, or adolescent at the time of the transplant.

[0050] In certain embodiments, methods and systems involving the transplantation of autologous thymic tissue from a newborn or infant into the lymph nodes of a subject when the subject is a newborn, infant, or child are advantageous compared to the transplantation of fetal or adult thymic tissue into an adult subject. While not wishing to be bound by any particular theory, the methods and systems of the present invention are advantageous because, among other advantages, transplanting autologous neonatal thymic tissue into the lymph nodes of a young subject (e.g., a newborn, infant, or child) can provide the transplanted thymocytes with an environment for growth and function similar to that in which the innate neonatal thymus grows and develops. Newborn mammals (e.g., humans) may have a limited peripheral immune system immediately after birth, with few T lymphocytes present in peripheral tissues. Therefore, the innate thymus may need to continue expanding to provide lymphocytes (e.g., naive T lymphocytes) to the peripheral immune system. During this early neonatal developmental process, the innate thymus can undergo dramatic changes at the molecular and cellular levels due to various influences from the thymus itself and the maturing subject, including the development of the subject's hormonal and immune systems. Unique molecular and cellular characteristics of neonatal thymic tissue have been reported. For example, gamma / delta T cells generated during fetal / neonatal thymic development may fail to develop or may develop at a reduced level in the adult thymus (Ito et al., Proc. Natl. Acad. Sci. USA, (1989) 86:631). Furthermore, a distinct population of regulatory T cells (Tregs) is produced in the perinatal thymus. These specific Tregs can play an essential role in maintaining self-tolerance and can be distinguished from adult-derived Tregs based on their molecular and activation profiles (Yang et al., Science. 2015 May 1;348(6234):589-594). As another example, CD4+ T cells derived from the neonatal thymus can have different functional properties than cells derived from the adult thymus (Becky Adkins, The Journal of Immunology, 2003, 171:5157-5164).Such unique developmental environment and changes in the neonatal thymus during early childhood may not be present at other developmental stages and may still be important for the structural and functional maturation of the thymus. As a result, the methods and systems of the present invention can provide a developmental environment that may not be present at other stages of a subject's life (e.g., when the subject is an adult). The methods and systems disclosed herein can be advantageous because, among other benefits, neonatal thymic tissue can develop compared to fetal thymic tissue and may have growth potential compared to adult thymic tissue that may have undergone age-related degeneration. Thus, neonatal thymic tissue may have a more favorable machinery for developing into ectopic thymic tissue with sufficient mass and function to restore the development and maturation of the immune system (e.g., naive T lymphocytes) in a subject.

[0051] In certain embodiments, the expansion of thymic tissue includes the engraftment, proliferation, differentiation, and / or growth of thymic tissue in the lymph node to produce ectopic thymic tissue in the lymph node. The produced ectopic thymic tissue is biologically active and has thymic function. In certain embodiments, the thymic tissue expands in the lymph node such that the mass of the ectopic tissue ultimately produced is greater than the original mass of thymic tissue delivered to the lymph node. In certain embodiments, the mass of ectopic tissue ultimately produced is at least about 1.1 times, 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 500 times, or at least about 1000 times the original mass of thymic tissue delivered to the lymph node.

[0052] In certain embodiments, angiogenesis can occur in lymph nodes that receive thymic tissue (e.g., there can be blood vessels that infiltrate into lymph nodes and into ectopic thymic tissue to form a vasculature network). In certain embodiments, the infiltrating vasculature network can form the basis of a blood supply to ectopic thymic tissue. In certain embodiments, the infiltrating vasculature network can assist in the transport of T cells to and from ectopic thymic tissue. In certain embodiments, the lymphatic circulatory system also serves as a transport channel for substances (e.g., T cells) produced by ectopic tissue. Thus, the lymphatic system, in certain embodiments, can provide such substances to the blood circulatory system through crosstalk with other parts of the body.

[0053] In certain embodiments, the amount of thymus tissue delivered to a subject (e.g., an effective amount as disclosed herein) is at least about 0.1 grams, at least about 0.2 grams, at least about 0.3 grams, at least about 0.4 grams, at least about 0.5 grams, at least about 0.6 grams, at least about 0.7 grams, at least about 0.8 grams, at least about 0.9 grams, at least about 1 gram, at least about 1.5 grams, at least about 2 grams, at least about 2.5 grams, at least about 3 grams, at least about 4 grams, at least about 5 grams, at least about 6 grams, at least about 7 grams, at least about 8 grams, at least about 9 grams, or at least about 10 grams. In certain embodiments, the amount of thymus tissue delivered to a subject (e.g., an effective amount as disclosed herein) is up to about 5 grams, up to about 10 grams, up to about 15 grams, or up to about 20 grams. In certain embodiments, the amount of thymus tissue delivered to a subject (e.g., an effective amount as disclosed herein) is about 0.1 grams, about 0.2 grams, about 0.3 grams, about 0.4 grams, about 0.5 grams, about 0.6 grams, about 0.7 grams, about 0.8 grams, about 0.9 grams, about 1 gram, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about 6 grams, about 8 grams, about 10 grams, about 13 grams, about 15 grams, about 18 grams, about 20 grams, or more. The weight can be measured before processing (e.g., before the thymus tissue is minced and added to a liquid to form a suspension). In some embodiments, the weight is measured after processing (e.g., after the thymus tissue is minced and added to a liquid to form a suspension). As disclosed herein, the aforementioned amount of thymus tissue can be delivered to a single lymph node or can be divided and delivered among two or more lymph nodes. As disclosed herein, the aforementioned amount of thymus tissue can be administered in a single dose or in two or more divided doses administered over any suitable period of time. In the case of two or more divided doses, the aforementioned amount can be the sum of the two or more divided doses (i.e., the amount per dose).

[0054] In some embodiments, thymus tissue is delivered to a subject in an amount relative to the subject's body weight (e.g., an effective amount as disclosed herein). For example, thymus tissue is delivered to a subject in an amount of at least about 0.001 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 15 mg / kg, at least about 20 mg / kg, at least about 25 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, at least about 50 mg / kg, at least about 60 mg / kg, at least about 70 mg / kg, at least about 80 mg / kg, at least about 90 mg / kg, at least about 100 mg / kg, at least about 150 mg / kg, at least about 20 mg / kg, at least about 25 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, at least about 50 mg / kg, at least about 60 mg / kg, at least about 70 mg / kg, at least about 80 mg / kg, at least about 100 mg / kg, at least about 150 mg / kg, at least about 20 mg / kg, at least about 25 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, at least about 50 mg / kg, at least about 60 mg / kg, at least about 70 mg / kg, at least about 100 mg / kg, at least about 150 mg / kg, at least about 150 mg / kg, at least about 150 mg / kg, at least about 150 mg / kg, at least about 150 mg / g / kg, at least about 90 mg / kg, at least about 100 mg / kg, at least about 110 mg / kg, at least about 120 mg / kg, at least about 130 mg / kg, at least about 140 mg / kg, at least about 150 mg / kg, at least about 160 mg / kg, at least about 170 mg / kg, at least about 180 mg / kg, at least about 190 mg / kg, at least about 200 mg / kg, at least about 250 mg / kg, at least about 500 mg / kg, at least about 1000 mg / kg, or at least about 1500 mg / kg. In some embodiments, thymus tissue is delivered to a subject in an amount of up to about 1 mg / kg, up to about 10 mg / kg, up to about 20 mg / kg, up to about 50 mg / kg, up to about 100 mg / kg, up to about 110 mg / kg, up to about 120 mg / kg, up to about 130 mg / kg, up to about 140 mg / kg, up to about 150 mg / kg, up to about 160 mg / kg, up to about 170 mg / kg, up to about 180 mg / kg, up to about 190 mg / kg, up to about 200 mg / kg, up to about 250 mg / kg, up to about 500 mg / kg, up to about 1000 mg / kg, or up to about 1500 mg / kg.In some embodiments, the thymus tissue is administered at about 0.001 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 140 mg / kg, about 150 mg / kg, about 160 mg / kg, about 170 mg / kg, about 180 mg / kg, about 190 mg / kg, about 210 mg / kg, about 220 mg / kg, about 230 mg / kg, about 240 mg / kg, about 250 mg / kg, about 260 mg / kg, about 270 mg / kg, about 280 mg / kg, about 290 mg / kg, about 300 mg / kg, about 310 mg / kg, about 320 mg / kg, about 330 mg / kg, about 340 mg / kg, about 350 mg / kg, about 360 mg / kg, about 370 mg / kg, about 380 mg / kg, about 390 mg / kg, about 400 mg / kg, about 410 mg / kg, about 420 mg / kg, about 430 mg / kg, about 440 mg / kg, about 450 mg / kg, about 460 mg / kg, about 470 mg / kg, about 480 mg / kg, about 490 mg / kg, about 50 The effective amount of thymus tissue is delivered to a subject in an amount of about 90 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, about 150 mg / kg, about 160 mg / kg, about 170 mg / kg, about 180 mg / kg, about 190 mg / kg, about 200 mg / kg, about 250 mg / kg, about 500 mg / kg, about 1000 mg / kg, or about 1500 mg / kg. The effective amount disclosed herein may be an amount relative to the body weight of the subject. The amount of thymus tissue can be measured before processing (e.g., before mincing the thymus tissue and adding it to a liquid to form a suspension). As disclosed herein, the amount of thymus tissue can be delivered to a single lymph node or can be divided among two or more lymph nodes. As disclosed herein, a given size of thymus tissue can be administered in a single dose or in two or more divided doses administered over any suitable period of time. In the case of two or more divided doses, the amount of thymus tissue can be the sum of the two or more divided doses (i.e., the amount per dose).

[0055] In certain embodiments, the size of the thymus tissue delivered into the subject is at least about 0.1 cm 3 , at least about 0.2 cm 3 , at least about 0.3 cm 3 , at least about 0.4 cm 3 , at least about 0.5 cm 3 , at least about 0.6 cm 3 , at least about 0.7 cm 3 , at least about 0.8 cm 3 , at least about 0.9 cm 3 , at least about 1 cm 3, at least about 1.5 cm 3 , at least about 2 cm 3 , at least about 2.5 cm 3 , or at least about 3 cm 3 In certain embodiments, the size of the thymus tissue delivered into the subject is about 5 cm 3 Up to about 10cm 3 Up to about 15cm 3 up to, or about 20 cm 3 In certain embodiments, the size of the thymus tissue delivered into the subject is up to about 0.1 cm 3 , about 0.2 cm 3 , about 0.3cm 3 , about 0.4cm 3 , about 0.5cm 3 , about 0.6 cm 3 , about 0.7cm 3 , about 0.8cm 3 , about 0.9cm 3 , about 1cm 3 , about 2 cm 3 , about 3cm 3 , about 4cm 3 , about 5cm 3 , about 6 cm 3 , about 8cm 3 , about 10cm 3 , about 13cm 3 , about 15cm 3 , about 18cm 3 , about 20cm 3or more. The effective amount disclosed herein can be expressed in terms of size. Size can be measured before processing (e.g., before the thymus tissue is minced and added to a liquid to form a suspension). In some embodiments, size is measured after processing (e.g., after the thymus tissue is minced and added to a liquid to form a suspension). As disclosed herein, thymus tissue of a given size can be delivered to one lymph node or can be split and delivered among two or more lymph nodes. As disclosed herein, thymus tissue of a given size can be administered in a single dose, or in two or more doses administered at any suitable time period. In the case of two or more doses, the thymus tissue of a given size can be the sum of the two or more doses (i.e., the amount per dose).

[0056] In certain embodiments, the thymus tissue delivered to a subject is in the form of a liquid suspension. In some embodiments, the volume of the thymus tissue delivered to a subject in liquid suspension is at least about 0.1 mL, at least about 0.2 mL, at least about 0.3 mL, at least about 0.4 mL, at least about 0.5 mL, at least about 0.6 mL, at least about 0.7 mL, at least about 0.8 mL, at least about 0.9 mL, at least about 1 mL, at least about 1.5 mL, at least about 2 mL, at least about 2.5 mL, at least about 3 mL, at least about 4 mL, at least about 5 mL, at least about 6 mL, at least about 7 mL, at least about 8 mL, at least about 9 mL, or at least about 10 mL (e.g., volume per lymph node, or total volume of divided volumes into two or more lymph nodes). In certain embodiments, the volume of thymic tissue delivered into a subject is up to about 5 mL, up to about 10 mL, up to about 15 mL, or up to about 20 mL (e.g., divided for delivery to two or more lymph nodes). In certain embodiments, the volume of thymic tissue delivered into a subject is about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 2 mL, or about 3 mL per lymph node. In certain embodiments, the volume of thymus tissue delivered into a subject is about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 8 mL, about 10 mL, about 13 mL, about 15 mL, about 18 mL, about 20 mL, or more (divided for delivery to two or more lymph nodes). As disclosed herein, the aforementioned volume can be administered in a single dose or divided into two or more doses and administered over any suitable period of time. In the case of two or more doses, the volume can be the sum of the volumes divided into two or more doses (i.e., the volume per dose). The effective amount disclosed herein can be expressed in terms of volume.

[0057] The thymus tissue can be administered to a subject in a single dose or in two or more divided doses over any suitable period of time, including multiple doses at regular or irregular intervals, such as about half an hour, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 16 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 1 month, about 1 month, about 2 weeks, about 3 months, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 1 month, about 1 month, about 2 weeks, about 3 weeks ...1 month, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 3 weeks, about 4 weeks, about 4 weeks, about 5 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 3 weeks, about 4 weeks, about 4 The administration can be divided into two doses at 0 weeks, about 11 weeks, about 12 weeks, about 3 months, about 16 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 14 months, about 16 months, about 18 months, about 20 months, about 22 months, about 24 months, about 30 months, about 36 months, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. In some embodiments, thymus tissue can be administered to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times.

[0058] In certain embodiments, the disclosed subject matter can increase the number of circulating T cells in a subject's blood and restore normal immune function. In a non-limiting embodiment, at least about one-third of a subject's total thymus weight can be implanted to increase the number of circulating T cells in the blood and restore normal immunity. For example, more than about 3 mg of thymus tissue can be delivered to a mouse subject, where the standard size of a newborn thymus averages about 8.5 mg. In human subjects, about 7 grams (gr), about 8 gr, about 9 gr, or about 10 gr of thymus tissue can be delivered to a human subject, where the standard size of a childhood thymus averages 25 gr. The total thymus weight can be the standard weight for a newborn thymus. The total thymus weight can be the standard weight for an infant thymus. The total thymus weight can be the standard weight for a child's thymus. The total thymus weight can be the standard weight for a child's thymus. The total thymus weight can be the standard weight for an adult thymus. In some embodiments, the total thymus weight can be the total thymus weight of a normal (e.g., healthy or immunocompetent) individual of a similar age to the subject, hi some embodiments, the total thymus weight can be the total thymus weight of a normal individual of a different age than the subject.

[0059] In certain embodiments, the disclosed subject matter can increase circulating CD3+ T cells in a subject and restore the naive T cell population. For example, a subject may suffer from lymphopenia, which can reduce naive T cells accompanied by a reduction in memory T cell populations. Quantitative deficiencies in the T cell compartment can also affect the naive T cell population. The disclosed subject matter can restore the naive T cell population in such a subject by increasing circulating CD3+ T cells. In some embodiments, the disclosed subject matter can reduce T cell senescence in a subject.

[0060] In some embodiments, the frequency of CD45+ peripheral blood cells that are T cells in a subject can be increased or maintained by the compositions and methods disclosed herein. For example, in some instances, the frequency of CD45+ peripheral blood cells that are T cells can be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% after delivery of thymic tissue to a lymph node as disclosed herein. In some embodiments, the frequency of CD45+ peripheral blood cells that are T cells can be increased by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or more, compared to before delivery of thymic tissue as disclosed herein or compared to a comparable subject that did not receive thymic tissue. For example, the frequency of CD45+ peripheral blood cells that are T cells can be 1% before delivery of the thymic tissue disclosed herein and can increase by 10% after delivery of the thymic tissue to achieve a frequency of 11%.

[0061] In some embodiments, the concentration of T cells in the peripheral blood of a subject can be increased by the compositions and methods disclosed herein. For example, in some instances, the concentration of T cells in the peripheral blood of a subject can be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, at least about 1000-fold, or at least about 10,000-fold or more, e.g., compared to before delivery of the thymic tissue disclosed herein or compared to a comparable subject that has not received thymic tissue. Concentration can be expressed as, for example, cells per cubic millimeter, microliter, deciliter, milliliter, or liter.

[0062] Methods for identifying T cells are known in the art. In some embodiments, T cells are identified based on being CD3+, CD3+CD4+, CD3+CD8+, CD3+CD4+CD45+, or CD3+CD8+CD45+.

[0063] In some embodiments, the frequency of CD3+ peripheral blood cells that are naive T cells can be increased or maintained in a patient by the methods disclosed herein. For example, in some instances, the frequency of CD3+ peripheral blood cells that are naive T cells can be at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% after delivery to thymic tissue to lymph nodes as disclosed herein. In some embodiments, the frequency of CD3+ peripheral blood cells that are naive T cells can be increased by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or more, compared to before delivery of the thymic tissue disclosed herein or compared to a comparable subject that has not received thymic tissue. For example, the frequency of CD3+ peripheral blood cells that are naive T cells can be 1% before delivery of the thymic tissue disclosed herein and can increase by 10% after delivery of the thymic tissue to achieve a frequency of 11%.

[0064] In some embodiments, the concentration of naive T cells in the peripheral blood of a subject can be increased by the compositions and methods disclosed herein. For example, in some instances, the concentration of naive T cells in the peripheral blood of a subject can be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, at least about 1000-fold, or at least about 10,000-fold or more, e.g., compared to before delivery of thymic tissue disclosed herein or compared to a comparable subject that has not received thymic tissue. Concentration can be expressed as, for example, cells per cubic millimeter, microliter, deciliter, milliliter, or liter.

[0065] Methods for identifying naive T cells, other subsets disclosed herein, and equivalent populations in other species (e.g., humans) are known in the art. In some embodiments, naive T cells are CD3+CD4+CD44-CD62L+ or CD3+CD8+CD44-CD62L+. In some embodiments, naive T cells are CD3+CD4+CD45RA+CCR7+, CD3+CD8+CD45RA+CCR7+, or any combination of CD3+, CD4+, CD8+, CD45RA+, CCR7+, CD45RO-, and CD27+.

[0066] In some embodiments, the compositions and methods of the present disclosure can be used to obtain a particular CD4:CD8 T cell ratio in a subject (e.g., the CD4:CD8 ratio of total circulating T cells, total circulating naive T cells, total circulating effector T cells, total circulating effector / memory T cells, total circulating central memory T cells, or a combination thereof). For example, the CD4:CD8 ratio obtained by the compositions and methods disclosed herein can be at least about 1:1, at least about 1.1:1, at least about 1.2:1, at least about 1.3:1, at least about 1.4:1, at least about 1.5:1, at least about 1.6:1, at least about 1.7:1, at least about 1.8:1, at least about 1.9:1, or at least about 2:1. In some embodiments, the CD4:CD8 ratio achieved by the compositions and methods disclosed herein can be at most about 2: 1, at most about 2.1: 1, at most about 2.2: 1, at most about 2.3: 1, at most about 2.4: 1, at most about 2.5: 1, at most about 2.6: 1, at most about 2.7: 1, at most about 2.8: 1, at most about 2.9: 1, at most about 3: 1, at most about 2.5: 1, at most about 3: 1, at most about 4: 1, or at most about 5: 1. In some embodiments, the CD4:CD8 ratio achieved by the compositions and methods disclosed herein is about 2: 1.

[0067] The effect of the compositions and methods of the present disclosure on the outcomes disclosed herein can be assessed at any suitable time after delivery of thymic tissue to the lymph nodes of a subject. For example, the effect on the number and / or frequency of peripheral blood T cells, the concentration and / or frequency of naive peripheral blood T cells, the CD4:CD8 ratio, lymph node weight, the expansion of thymic tissue in the lymph nodes, or any other outcome disclosed herein can be assessed at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 1 month, about 1 month, about 2 months, about 3 months, about 4 months, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 3 months, about 4 months, about 5 weeks, about 6 months, about 7 weeks, about 8 weeks, about 9 weeks, about 1 month, about 1 month, about 2 months, about 3 months, about 4 months, about 5 weeks, about 6 weeks ...1 month, about 2 months, about 3 months, about 4 months, about 5 weeks, about 1 month, about 2 months, about Evaluation can be performed after 0 weeks, about 11 weeks, about 12 weeks, about 3 months, about 16 weeks, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 14 months, about 16 months, about 18 months, about 20 months, about 22 months, about 24 months, about 30 months, about 36 months, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years.

[0068] Any method known in the art for delivering agents, materials, or cells to an internal organ of a subject can be used with the subject matter of the present disclosure. In certain embodiments, thymus tissue is delivered to a lymph node of a subject by a needle. In certain embodiments, the needle is a microinjection needle. In certain embodiments, the method disclosed herein includes introducing a needle into a lymph node of a subject. A minimally invasive procedure is performed under ultrasound guidance, which may have the operational advantage of being less traumatic to the body than open surgery. Furthermore, minimally invasive procedures can cause less pain, shorter hospital stays, and fewer complications than open surgery. In certain embodiments, the method disclosed herein is performed using a minimally invasive procedure. In certain embodiments, the minimally invasive procedure is performed under ultrasound guidance. Non-limiting examples of minimally invasive procedures that can be used include those disclosed in PCT Patent Application No. PCT / US2020 / 027783, the entire contents of which are incorporated herein by reference.

[0069] 6.2.2 Lymph nodes In certain embodiments, the thymus tissue is delivered into at least one lymph node of a subject. In certain embodiments, the thymus tissue is delivered into at least two, at least three, at least four, at least five, at least six, at least five, at least seven, at least eight, at least nine, at least ten, or more lymph nodes of a subject. In non-limiting embodiments, about 0.05 ml, about 0.1 ml, about 0.5 ml, about 1 ml, about 2 ml, or about 3 ml of thymus tissue can be delivered into the lymph node.

[0070] Non-limiting examples of lymph nodes to which thymus tissue can be delivered using the methods disclosed herein include abdominal lymph nodes, celiac lymph nodes, para-aortic lymph nodes, splenic hilar lymph nodes, hepatic hilar lymph nodes, left gastric lymph nodes, right gastric lymph nodes, left gastroepiploic (gastro-omental) lymph nodes, right gastroepiploic (gastro-omental) lymph nodes, retroperitoneal lymph nodes, pyloric lymph nodes (e.g., suprapyloric lymph nodes, infrapyloric lymph nodes, retropyloric lymph nodes), pancreatic lymph nodes (e.g., superior pancreatic lymph nodes, inferior pancreatic lymph nodes, splenic gland lymph nodes, lymph nodes), splenic lymph nodes, hepatic lymph nodes (e.g., gallbladder lymph nodes, omental foramen lymph nodes, foramen of Winslow), pancreaticoduodenal lymph nodes (e.g., superior pancreaticoduodenal lymph nodes, inferior pancreaticoduodenal lymph nodes), superior mesenteric lymph nodes, ileocolic lymph nodes, prececal lymph nodes, retrocecal lymph nodes, appendix lymph nodes, mesocolic lymph nodes (e.g., paracolic lymph nodes, left colonic lymph nodes, middle colonic lymph nodes, right colonic lymph nodes, inferior mesenteric lymph nodes, sigmoid colonic lymph nodes, upper rectal lymph nodes) lymph nodes), common iliac lymph nodes (e.g., medial common iliac lymph nodes, middle common iliac lymph nodes, lateral common iliac lymph nodes, subaortic common iliac lymph nodes, promontory common iliac lymph nodes), and external iliac lymph nodes (e.g., medial external iliac lymph nodes, middle external iliac lymph nodes, lateral external iliac lymph nodes, medial hiatal-femoral lymph nodes, middle hiatal-femoral lymph nodes, lateral hiatal-femoral lymph nodes, interiliac external iliac lymph nodes, obturator-external iliac-obturator lymph nodes).

[0071] In certain embodiments, lymph nodes present in the peritoneal cavity may be particularly useful, for example, when the lymph nodes are not closely associated with an artery or vena cava, as it is important that the lymph nodes be able to enlarge as the transplanted thymic tissue expands.

[0072] 6.2.3 Subjects In certain embodiments, a subject to be treated by the methods disclosed herein has undergone a thymectomy. In certain embodiments, a subject to be treated by the methods disclosed herein has thymus tissue delivered into the subject's lymph nodes during thymectomy. In certain embodiments, the subject has a congenital heart defect and has undergone or is scheduled to undergo open-heart surgery. Open-heart surgery has become a routine method of treating serious congenital heart defects. Early cardiac surgical intervention for congenital heart defects can involve thymectomy (e.g., partial or complete removal of the thymus). In certain embodiments, the subject underwent thymectomy during open-heart surgery. In certain embodiments, the subject underwent thymectomy when the subject was a newborn. In certain embodiments, the subject underwent thymectomy when the subject was an infant. In certain embodiments, the subject underwent thymectomy when the subject was a child. In certain embodiments, the thymectomy was performed when the subject was up to 1 month old, up to 2 months old, up to 3 months old, up to 4 months old, up to 5 months old, up to 6 months old, up to 8 months old, up to 10 months old, up to 1 year old, or up to 2 years old. In certain embodiments, the thymectomy was performed when the subject was up to 1 month old.

[0073] In some embodiments, a subject to be treated with the compositions and methods of the present disclosure has a condition affecting the thymus, including, but not limited to, myasthenia gravis, true erythroid aplasia, hypogammaglobulinemia, thymic carcinoma, thymoma, thymoma type A, thymoma type B, autoimmune disease, T-cell-mediated autoimmunity, T-cell lymphopenia, thymic atrophy, age-related thymic atrophy, thymic cysts, thymic hyperplasia, thymic hypoplasia, thymic aplasia, thymic dysplasia, severe combined immunodeficiency, Nezerov syndrome, Wiscott-Aldrich syndrome, DiGeorge syndrome, recurrent infections, recurrent viral infections, immunological premature aging, and cancer.

[0074] Subjects to be treated by the methods disclosed herein can be of any age, hi certain embodiments, subjects to be treated by the methods disclosed herein are neonates, infants, children, adolescents, or adults.

[0075] In certain embodiments, the subject to be treated by the methods disclosed herein is a human newborn who has reached the age of about 1 month. In certain embodiments, the subject to be treated by the methods disclosed herein is a human infant who has reached the age of about 1 month and about 2 years. In certain embodiments, the subject to be treated by the methods disclosed herein is a human child who has reached the age of about 2 years and about 12 years. In certain embodiments, the subject to be treated by the methods disclosed herein is a human adolescent who has reached the age of about 12 years and about 18 years. In certain embodiments, the subject to be treated by the methods disclosed herein is a human adult who has reached the age of at least about 18 years. In certain embodiments, the subject to be treated is a middle-aged or elderly human (e.g., with decreased thymic function due to aging).

[0076] In certain embodiments, the subject to be treated by the methods disclosed herein is a human subject who is up to about 1 month old, up to about 3 months old, up to about 6 months old, up to about 1 year old, up to about 2 years old, up to about 3 years old, up to about 4 years old, up to about 5 years old, up to about 6 years old, up to about 7 years old, up to about 8 years old, up to about 9 years old, up to about 10 years old, up to about 11 years old, up to about 12 years old, up to about 13 years old, up to about 14 years old, up to about 15 years old, up to about 16 years old, up to about 17 years old, up to about 18 years old, or has reached at least 18 years of age.

[0077] In certain embodiments, the subject to be treated by the methods disclosed herein is a human subject who has reached the age of at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, at least about 12 years, at least about 13 years, at least about 14 years, at least about 15 years, at least about 16 years, at least about 17 years, at least about 18 years, at least about 25 years, at least about 30 years, at least about 35 years, at least about 40 years, at least about 45 years, at least about 50 years, at least about 55 years, at least about 60 years, at least about 65 years, at least about 70 years, or at least about 75 years of age.

[0078] In certain embodiments, thymus tissue is obtained from a subject when the subject is a newborn, infant, or child, and the thymus tissue is cryopreserved and then thawed and delivered to the subject. In certain embodiments, the cryopreserved thymus tissue can be delivered to a subject of any age after thymectomy. In certain embodiments, the thymus tissue obtained from a subject can be cryopreserved in multiple vials, each vial containing a single dose of thymus tissue that can be delivered to the subject. In certain embodiments, the cryopreserved thymus tissue is delivered to the subject multiple times over the course of the subject's lifetime.

[0079] 6.3 Kits The present disclosure provides kits for protecting, restoring, or enhancing thymic function in a subject (e.g., a subject who has undergone or will undergo thymectomy). In certain embodiments, the kit includes thymic tissue and tools for delivery of the thymic tissue to the subject, wherein the thymic tissue is autologous to the subject. In certain embodiments, the thymic tissue is provided in a solution included in the kit.

[0080] In certain embodiments, the solution contains different cell types that make up thymus tissue. In certain embodiments, the solution further comprises a pharmaceutically acceptable excipient, diluent, or carrier. Pharmaceutically acceptable carriers and diluents include saline, buffers, solvents, and / or dispersion media. Non-limiting examples of pharmaceutically acceptable excipients that can be used with the kits of the present disclosure include sugars (such as lactose, glucose, and sucrose); starches (such as corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate); powdered tragacanth; malt; gelatin; lubricants (such as magnesium stearate, sodium lauryl sulfate, and talc); cocoa butter and suppository wax; oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil); glycols. These include: alcohols (such as propylene glycol); polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG)); esters (such as ethyl oleate and ethyl laurate); agar; buffers (such as magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH-adjusted buffers; polyesters, polycarbonates, and / or polyanhydrides; bulking agents (such as polypeptides and amino acids); serum components (such as serum albumin, HDL, and LDL); C2-C12 alcohols (such as ethanol); and other non-toxic compatible substances used in pharmaceutical formulations.

[0081] In certain embodiments, the thymus tissue contained in the kit is cryopreserved. In certain embodiments, the thymus tissue contained in the kit is cryopreserved for about 12 hours, about 24 hours, about 2 days, about 3 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, or more. In certain embodiments, the thymus tissue included in the kit is cryopreserved for at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 8 months, at least about 10 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more. In certain embodiments, the thymus tissue included in the kit is cryopreserved for up to about 12 hours, up to about 24 hours, up to about 2 days, up to about 3 days, up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 8 months, up to about 10 months, up to about 1 year, up to about 2 years, up to about 3 years, up to about 4 years, up to about 5 years, up to about 6 years, up to about 7 years, up to about 8 years, up to about 9 years, up to about 10 years, up to about 15 years, up to about 20 years, up to about 30 years, up to about 40 years, up to about 50 years, or less.

[0082] In certain embodiments, the amount of thymus tissue included in the kit is at least about 0.1 gram, at least about 0.2 gram, at least about 0.3 gram, at least about 0.4 gram, at least about 0.5 gram, at least about 0.6 gram, at least about 0.7 gram, at least about 0.8 gram, at least about 0.9 gram, at least about 1 gram, at least about 1.5 gram, at least about 2 gram, at least about 2.5 gram, at least about 3 gram, at least about 4 gram, at least about 5 gram, at least about 6 gram, at least about 7 gram, at least about 8 gram, at least about 9 gram, or at least about 10 gram. In certain embodiments, the amount of thymus tissue included in the kit is up to about 5 gram, up to about 10 gram, up to about 15 gram, or up to about 20 gram. In certain embodiments, the amount of thymus tissue included in the kit is about 0.1 grams, about 0.2 grams, about 0.3 grams, about 0.4 grams, about 0.5 grams, about 0.6 grams, about 0.7 grams, about 0.8 grams, about 0.9 grams, about 1 gram, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about 6 grams, about 8 grams, about 10 grams, about 13 grams, about 15 grams, about 18 grams, about 20 grams, or more.

[0083] In certain embodiments, the size of the thymus tissue included in the kit is at least about 0.1 cm 3 , at least about 0.2 cm 3 , at least about 0.3 cm 3 , at least about 0.4 cm 3 , at least about 0.5 cm 3 , at least about 0.6 cm 3 , at least about 0.7 cm 3 , at least about 0.8 cm 3 , at least about 0.9 cm 3 , at least about 1 cm 3 , at least about 1.5 cm 3 , at least about 2 cm 3 , at least about 2.5 cm 3 , at least about 3 cm 3 At least about 4 cm 3 , or at least about 5 cm3 In certain embodiments, the size of the thymus tissue included in the kit is about 5 cm 3 Up to about 10cm 3 Up to about 15cm 3 up to, or about 20 cm 3 In certain embodiments, the size of the thymus tissue included in the kit is up to about 0.1 cm 3 , about 0.2 cm 3 , about 0.3cm 3 , about 0.4cm 3 , about 0.5cm 3 , about 0.6 cm 3 , about 0.7cm 3 , about 0.8cm 3 , about 0.9cm 3 , about 1cm 3 , about 2 cm 3 , about 3cm 3 , about 4cm 3 , about 5cm 3 , about 6 cm 3 , about 8cm 3 , about 10cm 3 , about 13cm 3 , about 15cm 3 , about 18cm 3 , about 20cm 3 , or even more.

[0084] In certain embodiments, the amount of thymic tissue included in the kit is an amount effective to restore thymic function in a subject. In certain embodiments, the amount of thymic tissue included in the kit is an amount effective to expand in a lymph node, where the expanded thymic tissue restores thymic function in the subject. In certain embodiments, the amount of thymic tissue included in the kit is an amount effective to increase thymic function in a subject. In certain embodiments, the amount of thymic tissue included in the kit is an amount effective to engraft into a lymph node. In certain embodiments, the amount of thymic tissue included in the kit is an amount effective to form ectopic thymic tissue in a lymph node. In certain embodiments, the amount of thymic tissue included in the kit is an amount effective to increase the level of circulating T cells (e.g., circulating naive T cells) in a subject. In certain embodiments, the amount of thymic tissue included in the kit is an amount effective to increase the density of circulating T cells in a subject (e.g., increasing the TCR repertoire and recognized alloantigens).

[0085] In some embodiments, the kits of the present disclosure can include components suitable for harvesting thymic tissue, processing thymic tissue, storing (e.g., cryopreserving) thymic tissue, culturing thymic tissue, delivering thymic tissue to a subject, or combinations thereof, hi certain embodiments, the kit does not include thymic tissue.

[0086] In certain embodiments, the kit includes materials (e.g., reagents and / or containers) necessary to cryopreserve thymus tissue. In certain embodiments, the kit is intended to cryopreserve thymus tissue for about 12 hours, about 24 hours, about 2 days, about 3 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, about 20 years, or more. In certain embodiments, the kit is intended for cryopreserving thymus tissue for at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 8 months, at least about 10 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 15 years, at least about 20 years, or more. In certain embodiments, the kit is intended for cryopreserving thymus tissue for up to about 12 hours, up to about 24 hours, up to about 2 days, up to about 3 days, up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months, up to about 8 months, up to about 10 months, up to about 1 year, up to about 2 years, up to about 3 years, up to about 4 years, up to about 5 years, up to about 6 years, up to about 7 years, up to about 8 years, up to about 9 years, up to about 10 years, up to about 15 years, up to about 20 years, up to about 30 years, up to about 40 years, up to about 50 years, or less.

[0087] In certain embodiments, the kit is intended for the collection, processing, storage, cultivation, and / or delivery of thymus tissue in an amount of at least about 0.1 gram, at least about 0.2 gram, at least about 0.3 gram, at least about 0.4 gram, at least about 0.5 gram, at least about 0.6 gram, at least about 0.7 gram, at least about 0.8 gram, at least about 0.9 gram, at least about 1 gram, at least about 1.5 gram, at least about 2 gram, at least about 2.5 gram, at least about 3 gram, at least about 4 gram, at least about 5 gram, at least about 6 gram, at least about 7 gram, at least about 8 gram, at least about 9 gram, or at least about 10 gram.

[0088] In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of thymic tissue in amounts of up to about 5 grams, up to about 10 grams, up to about 15 grams, or up to about 20 grams. In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of thymic tissue in amounts of about 0.1 grams, about 0.2 grams, about 0.3 grams, about 0.4 grams, about 0.5 grams, about 0.6 grams, about 0.7 grams, about 0.8 grams, about 0.9 grams, about 1 gram, about 2 grams, about 3 grams, about 4 grams, about 5 grams, about 6 grams, about 8 grams, about 10 grams, about 13 grams, about 15 grams, about 18 grams, about 20 grams, or more.

[0089] In certain embodiments, the kit comprises at least about 0.1 cm 3 , at least about 0.2 cm 3 , at least about 0.3 cm 3 , at least about 0.4 cm 3 , at least about 0.5 cm 3 , at least about 0.6 cm 3 , at least about 0.7 cm 3 , at least about 0.8 cm 3 , at least about 0.9 cm 3 , at least about 1 cm 3 , at least about 1.5 cm 3 , at least about 2 cm 3, at least about 2.5 cm 3 , at least about 3 cm 3 At least about 4 cm 3 , or at least about 5 cm 3 In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of thymus tissue in a size range of about 5 cm. 3 Up to about 10cm 3 Up to about 15cm 3 up to, or about 20 cm 3 In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of thymus tissue having a size up to about 0.1 cm. 3 , about 0.2 cm 3 , about 0.3cm 3 , about 0.4cm 3 , about 0.5cm 3 , about 0.6 cm 3 , about 0.7cm 3 , about 0.8cm 3 , about 0.9cm 3 , about 1cm 3 , about 2 cm 3 , about 3cm 3 , about 4cm 3 , about 5cm 3 , about 6 cm 3 , about 8cm 3 , about 10cm 3 , about 13cm 3 , about 15cm 3 , about 18cm 3 , about 20cm 3 The present invention is directed to the collection, processing, storage, culture, and / or delivery of thymic tissue having a size of or greater than 1000 mg / mL.

[0090] In some embodiments, the kit is intended for delivery of a volume of thymus tissue of at least about 0.1 mL, at least about 0.2 mL, at least about 0.3 mL, at least about 0.4 mL, at least about 0.5 mL, at least about 0.6 mL, at least about 0.7 mL, at least about 0.8 mL, at least about 0.9 mL, at least about 1 mL, at least about 1.5 mL, at least about 2 mL, at least about 2.5 mL, at least about 3 mL, at least about 4 mL, at least about 5 mL, at least about 6 mL, at least about 7 mL, at least about 8 mL, at least about 9 mL, or at least about 10 mL (e.g., volume per lymph node, or total volume of divided volumes to two or more lymph nodes). In some embodiments, the kit is intended for delivery of a volume of up to about 5 mL, up to about 10 mL, up to about 15 mL, or up to about 20 mL (e.g., divided for delivery to two or more lymph nodes). In some embodiments, the kit is intended for delivery of about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 2 mL, or about 3 mL of thymic tissue per lymph node. In some embodiments, the kit is intended for delivery of about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 8 mL, about 10 mL, about 13 mL, about 15 mL, about 18 mL, about 20 mL, or more of thymic tissue divided for delivery to two or more lymph nodes.

[0091] In some embodiments, the kit provides an amount of thymus tissue relative to the body weight of a subject (e.g., at least about 0.001 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 15 mg / kg, at least about 20 mg / kg, at least about 25 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, at least about 50 mg / kg, at least about 60 mg / kg, at least about 70 mg / kg, at least about 80 mg / kg, at least about 90 mg / kg, at least about 100 mg / kg, at least about 150 mg / kg, at least about 20 mg / kg, at least about 25 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, at least about 50 mg / kg, at least about 60 mg / kg, at least about 7 ...100 mg / kg, at least about 15 g / kg, at least about 80 mg / kg, at least about 90 mg / kg, at least about 100 mg / kg, at least about 110 mg / kg, at least about 120 mg / kg, at least about 130 mg / kg, at least about 140 mg / kg, at least about 150 mg / kg, at least about 160 mg / kg, at least about 170 mg / kg, at least about 180 mg / kg, at least about 190 mg / kg, at least about 200 mg / kg, at least about 250 mg / kg, at least about 500 mg / kg, at least about 1000 mg / kg, or at least about 1500 mg / kg) to the subject. In some embodiments, the amount is up to about 1 mg / kg, up to about 10 mg / kg, up to about 20 mg / kg, up to about 50 mg / kg, up to about 100 mg / kg, up to about 110 mg / kg, up to about 120 mg / kg, up to about 130 mg / kg, up to about 140 mg / kg, up to about 150 mg / kg, up to about 160 mg / kg, up to about 170 mg / kg, up to about 180 mg / kg, up to about 190 mg / kg, up to about 200 mg / kg, up to about 250 mg / kg, up to about 500 mg / kg, up to about 1000 mg / kg, or up to about 1500 mg / kg.In some embodiments, the amount is about 0.001 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg , about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 110 mg / kg, about 120 mg / kg, about 130 mg / kg, about 140 mg / kg, about 150 mg / kg, about 160 mg / kg, about 170 mg / kg, about 180 mg / kg, about 190 mg / kg, about 200 mg / kg, about 250 mg / kg, about 500 mg / kg, about 1000 mg / kg, or about 1500 mg / kg.

[0092] In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of an amount of thymic tissue effective to restore thymic function in a subject. In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of an amount of thymic tissue effective to expand in a lymph node, e.g., where the expanded thymic tissue restores thymic function in the subject. In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of an amount of thymic tissue effective to increase thymic function in a subject. In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of an amount of thymic tissue effective to engraft in a lymph node. In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of an amount of thymic tissue effective to form ectopic thymic tissue in a lymph node. In certain embodiments, the kit is intended for the collection, processing, storage, culture, and / or delivery of an amount of thymic tissue effective to increase the level of circulating T cells (e.g., circulating naive T cells) in a subject.

[0093] In certain embodiments, the kit further comprises instructions for protecting, enhancing, or restoring thymic function in a subject who has undergone or will undergo thymectomy, hi certain embodiments, the instructions comprise, e.g., a method described herein (e.g., in Section 6.2 of this disclosure).

[0094] Any suitable tool known in the art for delivering thymic tissue to a lymph node of a subject can be included in the kits disclosed herein. Non-limiting examples of tools for delivering thymic tissue include tubes, syringes, needles (e.g., glass needles for microinjection), containers suitable for cryopreservation, containers suitable for tissue culture, and any tools required for minimally invasive procedures.

[0095] While the inventive subject matter of this disclosure and certain advantages thereof have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of this disclosure. Moreover, the scope of this application is not intended to be limited to the particular embodiments of the processes, apparatus, manufacture, compositions, and methods described herein. As one skilled in the art will readily recognize from this disclosure of the inventive subject matter of this disclosure, existing or later-developed processes, apparatus, manufacture, compositions, or methods that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with the inventive subject matter of this disclosure. Accordingly, it is intended that the appended claims be included within the scope of such processes, apparatus, manufacture, compositions, or methods.

[0096] Various patents, patent applications, publications, product instructions, protocols, and sequence accession numbers are cited throughout this application, the disclosures of which are incorporated by reference herein in their entireties for all purposes. [Example]

[0097] 7. Working Example The inventive subject matter of the present disclosure will be better understood by reference to the following examples, which are provided as illustrations of the inventive subject matter of the present disclosure and do not limit the invention in any way.

[0098] 7.1 Example 1: Dosage Study in Nude Mice Nude mice lack a thymus and are therefore unable to produce T cells. As a result, they are immunodeficient. A functional immune system can be established in these mice by transplanting a thymus into the mesenteric lymph node. Here, we address the question of the amount of thymus tissue required to repopulate the blood with T cells and the amount of thymus tissue transplanted into the lymph node that will result in a sufficient number of circulating T cells.

[0099] Twenty-three 15-week-old BALB / c nude mice were enrolled (n = 16 females; n = 7 males). Twenty-one mice received transplants of 1 / 4 to 2 lobes of BALB / c wild-type neonatal thymus into the mouse lymph nodes (2 lobes = 1 whole thymus). The remaining two mice received transplants of a single thymic cell suspension from the same donor mouse. The samples to be transplanted were collected from 12 different thymuses in 2 liters, with a mean sample weight of 8.5 mg (range 6.2 to 11 mg; Figure 1A). Eight mice received transplants of 1 / 4 lobes of thymus, six mice received transplants of either 1 / 2 lobe or whole lobes of thymus, and one mouse received a whole thymus (2 lobes) (Figure 1B). When using the 1 / 2 lobe dose, the tissue was further fragmented into two pieces for transplantation. For the single-lobe dose, a total of four fragments were implanted into the lymph node, while for the two-lobe dose, a total of eight fragments were implanted. This approach was adopted because the small size of nude mouse lymph nodes (slightly over 1 mm on average) makes transplantation of tissue fragments difficult. Figure 1C shows the estimated implant weight of thymus fragments within the animals.

[0100] Four months after transplantation, 20 surviving mice were analyzed for the presence of T cells in their blood by flow cytometry. Three untransplanted BALB / c nude mice and three BALB / c wild-type mice served as negative and positive controls for circulating T cells, respectively. Using flow cytometry analysis, Figure 2A shows representative dot plots of CD3 expression under different conditions. Cells were first gated for singlets, then for live cells, CD45+ cells, and finally for CD3 expression. Letters indicate mouse IDs. The percentage of CD3+ T cells for each mouse is shown in Figure 2B, where round dots indicate females and square dots indicate males. All groups receiving at least one-quarter or more of the thymus transplant had increased proportions of circulating CD3+ T cells compared to non-transplanted controls. Mice transplanted with either a quarter or half lobe had similar percentages of CD3+ T cells (14.13 ± 3.29 and 11.76 ± 3.98, respectively; P = 0.2634). The percentage of CD3+ T cells was slightly higher in the single lobe compared with the half lobe (24.02 ± 7.43; P = 0.0067 for the half lobe vs. single lobe groups). The percentage of CD3+ T cells in the double lobe transplant was similar to the single lobe dose, whereas transplantation of a thymocyte cell suspension did not significantly increase the frequency of circulating T cells. Normal CD4 / CD8 ratios were obtained regardless of the amount of tissue transplanted (Figures 2C and 2D). Each dot represents a mouse. Circle dots represent females, and square dots represent males.

[0101] The percentage of CD3+ T cells in transplanted mice correlated with the actual weight (mg) of transplanted thymus tissue. The transplanted mice were divided into two groups: one containing mice transplanted with more than 1 mg to less than 3 mg of thymus tissue, and the other containing mice transplanted with more than 3 mg to less than 7 mg of thymus tissue. There was a statistically significant difference in the production of CD3+ blood T cells between these two groups (P = 0.0002; Figure 2E). In this analysis, mice were divided into two groups based on the actual weight (mg) of thymus tissue transplanted into their lymph nodes instead of thymic lobe fragments. *** indicates a P value less than 0.001.** indicates a P value less than 0.01. ns indicates "not significant" (i.e., a P value greater than 0.05).

[0102] These results suggest two conclusions: (1) there is a dose-response between the weight of thymic tissue transplanted into the lymph nodes and the percentage of circulating T cells in the transplanted mice, and (2) there is a threshold for the transplant weight of thymic tissue in the lymph nodes to increase the number of circulating T cells closer to wild-type (potentially improving T cell function).

[0103] One month later, the T cell receptor (TCR) Vβ repertoire of circulating T cells in five transplanted mice was analyzed by flow cytometry using monoclonal antibodies directed against 15 variants. The percentage of CD3+ T cells obtained at this time point was comparable to that obtained one month earlier, with the only exception being a slight increase in the percentage of T cells in mice transplanted with a whole thymus (Figure 3A). Averages were obtained from 16 separate measurements. Nine TCR-Vβ variants were identified as expressed in both the transplanted mice and one control BALB / c wild-type mouse (Figure 3B). BALB / c wild-type mice were included as a positive control (CNTRL). No differences were observed between the different conditions (one-way ANOVA P = 0.9956), demonstrating that delivery of thymic tissue into lymph nodes can increase the diversity of circulating T cells in recipients lacking a thymus or with a nonfunctional thymus.

[0104] Six and a half months after transplantation, all 20 mice were sacrificed. Figure 4A shows the weight of the isolated lymph nodes.

[0105] Similar to Figure 2B, the weight of lymph nodes transplanted with a quarter thymus lobe was similar to that of lymph nodes transplanted with a half thymus lobe (13.49 ± 7.65 and 18.33 ± 7.03, respectively; P = 0.2628; Figure 4A). Each dot represents a mouse. Circle dots represent females; square dots represent males. Lymph node weight was significantly heavier when a full thymus lobe was transplanted (28.8 ± 13.62; P = 0.0315 for the quarter lobe vs. full lobe groups). Using the same method as in Figure 2E, the isolated lymph nodes were divided into two groups based on the weight (mg) of transplanted thymus tissue. Again, there was a statistically significant difference between these two groups (P = 0.0428; Figure 4B). * indicates a P value less than 0.5.

[0106] The disclosed data suggest that there is a relationship between the weight of thymus tissue transplanted into the lymph nodes, the weight of the lymph nodes at the time of sacrifice, and the number of circulating T cells in the blood of transplanted mice. These results suggest that in size-limited subjects, more lymph nodes can be transplanted to obtain higher circulating T cell levels (e.g., a percentage closer to that of subjects with normally functioning thymuses). These results also suggest that females are more effective at generating new T cells after thymus transplantation in this model. Without wishing to be bound by theory, sex differences in thymus transplantation may be the result of hormonal differences between females and males in this model.

[0107] The above results indicate that increased thymus transplant weight can be associated with increased levels of naive T cell reconstitution. For example, in some embodiments, transplanting at least one-third of the total thymus weight can increase the level of naive T cell reconstitution compared to transplanting less than one-third of the total thymus weight. For example, more than 3 mg of a newborn mouse thymus, which has a standard size of an average of 8.5 mg, can be interpreted as slightly less than 9 grams of a childhood thymus, which has a standard size of 25 grams in a human patient. Considering that the maximum volume that can be injected into a human lymph node in some cases is approximately 1 ml, each of a total of nine lymph nodes can be injected (1 gram of thymus tissue per lymph node) to obtain one-third of an intact childhood thymus.

[0108] Thymectomy can result in T-cell lymphopenia, characterized by a decrease in naive T cells accompanied by an increase in the memory T-cell population. Furthermore, certain patients who undergo thymectomy (e.g., those in whom greater than 90% of the thymus is removed at an early age, such as less than 6 months of age) experience a quantitative deficiency in the T-cell compartment later in life, which primarily affects the naive T-cell population.

[0109] When these patients are given autologous thymic tissue transplants using the compositions and methods of the present disclosure, circulating CD3+ T cells can be increased, and in particular, naive T cell populations can be restored.

[0110] 7.2 Example 2: Study of Aged Wild-Type Mice This example examines whether transplantation of a low dose of thymus (1 / 4 lobe of thymus) into the lymph nodes can restore immune function in transplanted mice, and whether there are any differences between females and males.

[0111] Thirty-two C57BL / 6J mice (16 females and 16 males) were enrolled. Each mouse group consisted of eight adult mice (18 weeks old) and eight aged mice (62 weeks old). Half of the mice in each group received a quarter thymus transplant. Donor thymuses were isolated from GFP+ C57BL / 6J newborn mice from the same litter. Seven weeks after thymus transplantation, all 32 mice were analyzed by flow cytometry for naive, effector, and memory T cells, and differences in CD44 and CD62L expression in these subpopulations were investigated (Figures 5A-5H). The gating strategy is provided in Figures 5A-5H. Figure 5A shows the forward scatter gate versus the side scatter gate. Figure 5B shows the gate for single cells. Figure 5C shows the gate for live cells. Figure 5D shows the gate for CD45+ cells. Figure 5E shows gating for CD3+ cells. Figure 5F shows gating for CD4+ and CD8+ cells. Figure 5G shows gating for CD4+ cells for naive, activated effector, effector memory, and central memory cells. Figure 5H shows gating for CD8+ cells for naive, activated effector, effector, and central memory cells.

[0112] Naive T cells are continuously generated in the thymus, but as mice age and the thymus regresses, the proportions of naive CD4+ and CD8+ T cells decline. Therefore, we tested whether thymus transplantation in aged animals could increase the naive T cell population.

[0113] Aged female mice showed a statistically significant reduction in CD4+ naive T cells compared with adult female mice (P < 0.0001) (Figures 6A and 6E). The reduction in CD4+ naive T cells in these mice was accompanied by a corresponding significant reduction in activated CD4+ T cell effectors (P = 0.0062), CD4+ effector / memory (P < 0.0001), and CD4+ central memory T cells (P = 0.0297) (Figures 6A-6D). Cells were gated first for singlets, followed by live cells, CD45+ cells, CD3+ cells, CD4+ cells, and finally CD44 and CD62L expression (Figures 5A-5H). Each dot represents a mouse. Round dots represent females; square dots represent males. * indicates a P value less than 0.05. ** indicates a P value less than 0.01. *** indicates that P is less than 0.001. **** indicates a P value of less than 0.0001. Interestingly, aged male mice did not show any significant changes in these populations compared to adult male mice (Figures 6B-6E).

[0114] Importantly, CD4+ naive T cells in aged female mice were significantly increased after thymus transplantation compared to non-transplanted counterparts (P = 0.0127), whereas CD4+ effector / memory T cell populations were significantly decreased (P = 0.0321) (Figures 6A-6E). Furthermore, CD4+ effector / memory T cell populations were significantly decreased in aged male mice after thymus transplantation compared to non-transplanted counterparts (P = 0.0112) (Figures 6B-6E). No changes in CD4+ subpopulations were observed in either adult female or male mice after thymus transplantation (Figures 6B-6E).

[0115] These preliminary results indicate that (1) aged female mice have a lower percentage of naive T cells than age-matched male mice in this model; (2) aged female mice have a more pronounced decline in naive T cells than age-matched male mice in this model; and (3) in aged female mice, low-dose thymus transplantation increases the percentage of CD4+ naive T cells while decreasing the ratio of CD4+ effector / memory T cells.

[0116] 7.3 Example 3: Transplantation of Autologous Human Thymus Tissue A human neonatal or infant subject presents with an indication requiring thymectomy (e.g., a congenital heart defect requiring surgical correction and thymectomy as part of the surgery). Thymic tissue is harvested during surgery and cut or minced into small fragments using sterile techniques. If desired, the thymic tissue is cultured ex vivo and / or cryopreserved as disclosed herein. As disclosed herein, the thymic tissue fragments are suspended in a solution containing a suitable pharmaceutically acceptable excipient, diluent, or carrier and injected (e.g., using a minimally invasive procedure under ultrasound guidance) into one or more lymph nodes of the subject. One or several months later, a peripheral blood sample from the subject is evaluated to determine whether the levels of peripheral blood T cells and / or naive T cells are elevated compared to comparable subjects who did not receive the injection, or whether the levels are elevated compared to the levels of the subject before the fragment was injected. If the levels of peripheral blood T cells and / or naive T cells are still considered very low, more thymic tissue can be transplanted until sufficient numbers of peripheral blood T cells and / or naive T cells are obtained.

[0117] 7.4 Example 4: Transplantation of Thymus Tissue into Human Subjects The human subject presents with a condition affecting thymus function (e.g., age-related thymic atrophy). A suitable donor (e.g., a cadaveric donor with an intact thymus that is HLA-matched to the subject) is identified. Thymic tissue is harvested from the donor and cut or minced into small fragments using sterile techniques. Optionally, the thymic tissue is cultured ex vivo and / or cryopreserved as disclosed herein. As disclosed herein, the thymic tissue fragments are suspended in a solution containing a suitable pharmaceutically acceptable excipient, diluent, or carrier and injected (e.g., using a minimally invasive technique under ultrasound guidance) into one or more lymph nodes of the subject. If necessary, the subject is placed on an immunosuppressive regimen to improve graft acceptance. After one or several months, a peripheral blood sample from the subject is evaluated to determine whether the levels of peripheral blood T cells and / or naive T cells are elevated compared to comparable subjects who did not receive the injection, or compared to the levels in the subject before the fragment was injected. If the levels of peripheral blood T cells and / or naive T cells are still considered too low, more thymus tissue can be transplanted until a sufficient number of peripheral blood T cells and / or naive T cells are obtained.

[0118] While the inventive subject matter of this disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of this disclosure. Moreover, the scope of this application is not intended to be limited to the particular embodiments of the processes, apparatus, manufacture, compositions, and methods described herein. As one skilled in the art will readily recognize from disclosure of the inventive subject matter of this disclosure, existing or later-developed processes, apparatus, manufacture, compositions, or methods that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with the inventive subject matter of this disclosure. Accordingly, it is intended that the appended claims be included within the scope of such processes, apparatus, manufacture, compositions, or methods.

[0119] Various patents, patent applications, publications, product instructions, protocols, and sequence accession numbers are cited throughout this application, the disclosures of which are incorporated by reference herein in their entireties for all purposes. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for protecting or restoring thymic function in a subject in need thereof, comprising delivering thymic tissue into at least one lymph node of the subject, wherein the thymic tissue is autologous to the subject. (Item 2) Item 10. The method of item 1, wherein the subject has a congenital heart defect. (Item 3) 3. The method of claim 1 or 2, wherein the subject has undergone or is scheduled to undergo open heart surgery. (Item 4) 4. The method of any one of items 1 to 3, wherein the subject underwent thymectomy when he or she was a newborn or infant. (Item 5) 5. The method according to any one of items 1 to 4, wherein the subject is a human subject. (Item 6) 6. The method according to any one of items 1 to 5, wherein the subject is a newborn or an infant. (Item 7) 6. The method of any one of items 1 to 5, wherein the subject is a child, an adolescent, or an adult. (Item 8) 8. The method of any one of items 1 to 7, wherein the subject has undergone or is scheduled to undergo thymectomy. (Item 9) 9. The method of claim 8, wherein the thymus tissue is obtained from a subject undergoing thymectomy. (Item 10) 9. The method of any one of items 1 to 8, wherein the thymus tissue is obtained from a subject prior to thymectomy. (Item 11) 11. The method according to any one of items 1 to 10, wherein the thymus tissue is a minced thymus slice. (Item 12) 11. The method according to any one of items 1 to 10, wherein the thymus tissue is cultured ex vivo prior to the delivering step. (Item 13) 13. The method of claim 12, wherein the thymus tissue is cultured ex vivo for at least 24 hours prior to the delivering step. (Item 14) 14. The method of any one of items 1 to 13, wherein the thymus tissue is delivered into a lymph node of the subject by needle. (Item 15) 14. The method of any one of items 8 to 13, wherein the thymus tissue is delivered into the lymph node during the thymectomy. (Item 16) 14. The method according to any one of items 8 to 13, wherein the thymus tissue is delivered into the lymph node after the thymectomy. (Item 17) 17. The method of claim 16, wherein the thymus tissue is cryopreserved prior to delivery into the lymph nodes after the thymectomy. (Item 18) 18. The method of item 17, wherein the cryopreserved thymus tissue is delivered into a lymph node about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 4 months, about 6 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, about 15 years, about 20 years, or more after the thymectomy. (Item 19) 19. The method according to any one of items 1 to 18, wherein the amount of thymus tissue is an amount effective to restore thymus function in the subject. (Item 20) 20. The method of any one of items 1 to 19, wherein the amount of thymus tissue is effective to expand in the lymph node, and wherein the expanded thymus tissue restores thymic function in the subject. (Item 21) 21. The method of any one of items 1 to 20, wherein the amount of thymus tissue is at least about 0.1 grams. (Item 22) 22. The method according to any one of items 1 to 21, wherein the amount of thymus tissue is up to about 20 grams. (Item 23) The size of the thymus tissue is at least about 0.1 cm 3 23. The method according to any one of items 1 to 22, wherein (Item 24) The size of the thymus tissue is about 20 cm 3 24. The method according to any one of items 1 to 23, wherein (Item 25) 25. The method of any one of items 1 to 24, wherein the thymus tissue is delivered into at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 lymph nodes of the subject. (Item 26) 26. The method of any one of items 1 to 25, wherein at least about one-third of the subject's total thymus weight is delivered into at least one lymph node of the subject. (Item 27) 1. A kit for protecting or restoring thymic function in a subject, comprising thymic tissue and tools for delivering the thymic tissue to at least one lymph node of the subject, wherein the thymic tissue is autologous to the subject. (Item 28) 28. The kit of claim 27, further comprising a solution, wherein the thymus tissue is provided in the solution. (Item 29) 29. The kit of item 27 or 28, wherein the solution comprises a pharmaceutically acceptable excipient, a pharmaceutically acceptable diluent, or a pharmaceutically acceptable carrier. (Item 30) 30. The kit according to any one of items 27 to 29, wherein the amount of thymus tissue is an amount effective to restore thymus function in the subject. (Item 31) 31. The kit of any one of items 27 to 30, wherein the amount of thymus tissue is effective to expand in the lymph node, and wherein the expanded thymus tissue restores thymic function in the subject. (Item 32) 32. The kit of any one of items 27 to 31, further comprising instructions, wherein the instructions include delivering the thymus tissue to the at least one lymph node of the subject. (Item 33) 33. The kit of claim 32, wherein the instructions comprise delivering at least about one-third of the subject's total thymus weight into the at least one lymph node of the subject. (Item 34) 34. The kit of any one of items 27 to 33, wherein the tools for delivery of thymus tissue include needles and tools necessary for minimally invasive procedures. (Item 35) 35. The kit according to any one of items 27 to 34, wherein the thymus tissue is cryopreserved thymus tissue. (Item 36) 36. The kit of any one of items 27 to 35, wherein the subject has undergone or is scheduled to undergo thymectomy. (Item 37) A method comprising a step of delivering thymic tissue into a lymph node of a subject, wherein the frequency of CD45+ peripheral blood cells that are T cells in the subject is at least 20% after the step of delivering. (Item 38) 38. The method of item 37, wherein the frequency of CD45+ peripheral blood cells that are T cells is at least 25% after the delivering step. (Item 39) 38. The method of item 37, wherein the frequency of CD45+ peripheral blood cells that are T cells is at least 30% after the delivering step. (Item 40) 1. A method comprising delivering thymic tissue into a lymph node of a subject, wherein the frequency of CD45+ peripheral blood cells that are T cells in the subject is increased by at least 5% compared to the frequency of CD45+ peripheral blood cells that are T cells in the subject prior to the delivering step. (Item 41) 41. The method of claim 40, wherein the frequency of CD45+ peripheral blood cells that are T cells is increased by at least 10% compared to the frequency of CD45+ peripheral blood cells that are T cells in the subject before the delivering step. (Item 42) 41. The method of claim 40, wherein the frequency of CD45+ peripheral blood cells that are T cells is increased by at least 20% compared to the frequency of CD45+ peripheral blood cells that are T cells in the subject before the delivering step. (Item 43) 41. The method of claim 40, wherein the frequency of CD45+ peripheral blood cells that are T cells is increased by at least 30% compared to the frequency of CD45+ peripheral blood cells that are T cells in the subject before the delivering step. (Item 44) 1. A method comprising delivering thymic tissue into a lymph node of a subject, wherein the concentration of T cells in the peripheral blood of the subject is increased by at least 5% compared to the concentration of T cells in the peripheral blood of the subject before the delivering step. (Item 45) 45. The method of claim 44, wherein the concentration of T cells in the peripheral blood of the subject is increased by at least 10% compared to the concentration of T cells in the peripheral blood of the subject before the delivering step. (Item 46) 45. The method of claim 44, wherein the concentration of T cells in the peripheral blood of the subject is increased by at least 20% compared to the concentration of T cells in the peripheral blood of the subject before the delivering step. (Item 47) 45. The method of claim 44, wherein the concentration of T cells in the peripheral blood of the subject is increased by at least 30% compared to the concentration of T cells in the peripheral blood of the subject before the delivering step. (Item 48) A method comprising a step of delivering thymic tissue into a lymph node of a subject, wherein the frequency of CD3+ peripheral blood cells that are naive T cells in the subject is at least 20% after the delivering step. (Item 49) Item 49. The method of item 48, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is at least 25% after the delivering step. (Item 50) Item 49. The method of item 48, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is at least 30% after the delivering step. (Item 51) Item 49. The method of item 48, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is at least 40% after the delivering step. (Item 52) Item 49. The method of item 48, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is at least 50% after the delivering step. (Item 53) A method comprising a step of delivering thymic tissue into a lymph node of a subject, wherein the frequency of CD3+ peripheral blood cells that are naive T cells in the subject is increased by at least 5% compared to the frequency of CD3+ peripheral blood cells that are naive T cells in the subject before delivery. (Item 54) 54. The method of claim 53, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is increased by at least 10% compared to the frequency of CD3+ peripheral blood cells that are naive T cells in the subject before the delivering step. (Item 55) 54. The method of claim 53, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is increased by at least 20% compared to the frequency of CD3+ peripheral blood cells that are naive T cells in the subject before the delivering step. (Item 56) 54. The method of claim 53, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is increased by at least 30% compared to the frequency of CD3+ peripheral blood cells that are naive T cells in the subject before the delivering step. (Item 57) 54. The method of claim 53, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is increased by at least 40% compared to the frequency of CD3+ peripheral blood cells that are naive T cells in the subject before the delivering step. (Item 58) 54. The method of claim 53, wherein the frequency of CD3+ peripheral blood cells that are naive T cells is increased by at least 50% compared to the frequency of CD3+ peripheral blood cells that are naive T cells in the subject before the delivering step. (Item 59) 1. A method comprising delivering thymic tissue into a lymph node of a subject, wherein the concentration of naive T cells in the peripheral blood of the subject is increased by at least 5% compared to the concentration of naive T cells in the peripheral blood of the subject before the delivering step. (Item 60) 60. The method of claim 59, wherein the concentration of naive T cells in the peripheral blood of the subject is increased by at least 10% compared to the concentration of naive T cells in the peripheral blood of the subject before the delivering step. (Item 61) 60. The method of claim 59, wherein the concentration of naive T cells in the peripheral blood of the subject is increased by at least 20% compared to the concentration of naive T cells in the peripheral blood of the subject before the delivering step. (Item 62) 60. The method of claim 59, wherein the concentration of naive T cells in the peripheral blood of the subject is increased by at least 30% compared to the concentration of naive T cells in the peripheral blood of the subject before the delivering step. (Item 63) 60. The method of claim 59, wherein the concentration of naive T cells in the peripheral blood of the subject is increased by at least 40% compared to the concentration of naive T cells in the peripheral blood of the subject before the delivering step. (Item 64) 60. The method of claim 59, wherein the concentration of naive T cells in the peripheral blood of the subject is increased by at least 50% compared to the concentration of naive T cells in the peripheral blood of the subject before the delivering step. (Item 65) 65. The method of any one of items 48 to 64, wherein the naive T cells are CD45RA+CCR7+. (Item 66) 65. The method of any one of items 48 to 64, wherein the naive T cells are CD44-CD62L+ or their human equivalents. (Item 67) A method comprising the step of delivering at least about 7 grams of thymus tissue into one or more lymph nodes in a subject. (Item 68) 68. The method of any one of items 37 to 67, wherein at least 8 grams of thymus tissue is delivered into one or more lymph nodes in the subject. (Item 69) 68. The method of any one of items 37 to 67, wherein at least 9 grams of thymus tissue is delivered into one or more lymph nodes in the subject. (Item 70) 68. The method of any one of items 37 to 67, wherein at least 10 grams of thymus tissue is delivered into one or more lymph nodes in the subject. (Item 71) A method comprising the step of delivering thymus tissue into a lymph node of a subject, wherein the subject is at least 40 years of age. (Item 72) 72. The method of any one of items 37 to 71, wherein the subject is at least 50 years old. (Item 73) 72. The method of any one of items 37 to 71, wherein the subject is at least 55 years old. (Item 74) 72. The method of any one of items 37 to 71, wherein the subject is at least 60 years old. (Item 75) 72. The method of any one of items 37 to 71, wherein the subject is at least 65 years old. (Item 76) 72. The method of any one of items 37 to 71, wherein the subject is at least 70 years old. (Item 77) 1. A method comprising the step of delivering thymus tissue into a lymph node of a subject, wherein at least 50 milligrams of thymus tissue is delivered per kilogram of body weight of the subject. (Item 78) 78. The method of any one of items 37 to 77, wherein at least 100 milligrams of thymus tissue is delivered per kilogram of body weight of the subject. (Item 79) 79. The method according to any one of items 37 to 78, wherein the thymus tissue has been cryopreserved. (Item 80) 80. The method according to any one of items 37 to 79, wherein the thymus tissue has been cultured ex vivo. (Item 81) 81. The method of any one of items 37 to 80, wherein the thymus tissue is delivered in an amount effective to increase thymus function in the subject. (Item 82) 82. The method of any one of items 37 to 81, wherein the thymus tissue is delivered in an amount effective to form ectopic thymus tissue in the lymph node. (Item 83) 83. The method of any one of items 37 to 82, wherein the thymus tissue is delivered in an amount effective to increase T cell receptor density levels in the subject. (Item 84) 84. The method of any one of items 37 to 83, wherein the thymus tissue is delivered in a single dose. (Item 85) 84. The method of any one of items 37 to 83, wherein the thymus tissue is delivered in two or more doses. (Item 86) 86. The method of any one of items 37 to 85, wherein the thymus tissue is delivered to a single lymph node. (Item 87) 86. The method of any one of items 37 to 85, wherein the thymus tissue is delivered to two or more lymph nodes. (Item 88) 88. The method of any one of items 37 to 87, wherein the thymus tissue comprises minced thymus slices. (Item 89) 86. The method of any one of items 37 to 85, wherein the thymus tissue is injected into the lymph node. (Item 90) 90. The method of claim 89, wherein the thymus tissue is injected into the lymph node as part of a minimally invasive procedure. (Item 91) 91. The method of any one of items 37-70 or 77-90, wherein the subject is a newborn. (Item 92) 91. The method of any one of items 37-70 or 77-90, wherein the subject is an infant. (Item 93) 91. The method of any one of items 37-70 or 77-90, wherein the subject is a child. (Item 94) 91. The method of any one of items 37-70 or 77-90, wherein the subject is an adolescent. (Item 95) 91. The method of any one of items 37-70 or 77-90, wherein the subject is an adult. (Item 96) 91. The method of any one of items 37-70 or 77-90, wherein the subject is a mammal. (Item 97) 97. The method of any one of items 37 to 96, wherein the subject is a human. (Item 98) 98. The method of any one of items 37 to 97, wherein the subject has undergone thymectomy. (Item 99) 99. The method of claim 98, wherein the thymus tissue is obtained during a thymectomy. (Item 100) 99. The method of claim 98 or claim 99, wherein the thymus tissue is delivered to the lymph node during the thymectomy. (Item 101) 99. The method of claim 98 or claim 99, wherein the thymus tissue is delivered to the lymph node after the thymectomy. (Item 102) 102. The method of any one of items 37 to 101, wherein the subject has a condition affecting the thymus. (Item 103) 103. The method of claim 102, wherein the condition affecting the thymus is myasthenia gravis, true erythroid aplasia, hypogammaglobulinemia, thymic carcinoma, thymoma, type A thymoma, type B thymoma, autoimmune disease, T-cell mediated autoimmunity, T-cell lymphopenia, thymic atrophy, age-related thymic atrophy, thymic cysts, thymic hyperplasia, thymic hypoplasia, thymic aplasia, thymic dysplasia, severe combined immunodeficiency, Nezerov syndrome, Wiscott-Aldrich syndrome, DiGeorge syndrome, recurrent infections, recurrent viral infections, immunological premature aging, or cancer. (Item 104) 104. The method of any one of items 37 to 103, wherein the thymus tissue is autologous to the subject. (Item 105) 104. The method of any one of items 37 to 103, wherein the thymus tissue is allogeneic to the subject. (Item 106) 104. The method of any one of items 37 to 103, wherein the thymus tissue is HLA-matched to the subject. (Item 107) 107. The method of any one of items 37 to 106, further comprising administering to the subject an immunosuppressive regimen. (Item 108) 108. The method of any one of items 37 to 107, wherein the ratio of CD4 T cells:CD8 T cells in the subject's peripheral blood after the delivering step is between about 5:1 and 1:1.

Claims

[Claim 1] The invention described in the specification.

Images